PROJECTSUMMARY Voltage-gatedcalciumchannels(VGCC)regulatethefunctionofcellsrangingfromspermtoinsulin-producing pancreaticcellstoneurons.Thereisgrowingrecognitionthatspecificlipids,sterolsandthegangliosideGM1, regulateVGCCactivityinhealthanddiseaseincludingduringfertilization,regulationofbloodsugar,andpain sensation.However,determiningmechanismsbywhichlipidsregulateVGCCremainsamajorchallengeinthe field.Thisproposaladdressestwoquestionsofbroadimportancetobiology&medicine:Atamolecularlevel, howcanmembranelipidsregulate1)VGCCactivityand2)cellfunction?Spermareanoutstandingmodelfor theseissues,becausetheirabilitytofertilizeaneggiswell-knowntoberegulatedbylipiddynamics.However, thereistremendouscontroversyregardingspermcalcium(Ca2+)channels,withelectrophysiologicalrecordings (atroomtemperature)onlyidentifyingtheactivityofasingle,non-VGCCchannel(CatSper).Incontrast,data fromseveralgroupsusingapproachesofcellbiology,geneticsandpharmacology,showtheactivityofdifferent channelsincludingVGCC.Ofimportance,wefoundthatsterolsandGM1regulatespermVGCCactivityinways thattypicalelectrophysiologypracticescan?tdetect.Thisproposalrepresentsaconsortiumoftwolaboratories, onewithexpertiseinspermelectrophysiology,whohavepioneerednewmethodstobeabletodetectchannels regulatedbylipids.ThesecondlaboratorywasfirsttoidentifymembranelipidregulationofspermVGCC,and hasexpertiseinmembranelipidorganizationandfunction.Together,weproposetousenewtechnologiesand approachestoaddressthiscontroversy,whichiscentraltoourunderstandingofspermfunction&fertilization. Thisknowledgewillempowerclinicianstobetterunderstandthecausesofmaleinfertility,halfofwhicharedue tospermfunctiondefectsandarenotdetectedbytraditionalsemenanalysis.Basedonexcitingpreliminary datathatclearlyshowevidenceofmorethanonetypeofCa2+channel(usingspermfrommicenullfor CatSper),aswellaspublicationsfrombothlaboratories,weproposeaseriesofexperimentstoinvestigate howlipidsregulatemouseandhumanspermVGCCandpossiblyotherCa2+channels(Aim1).Thesestudies willutilizestate-of-the-artmicroscopy,pharmacology,mousegeneticmodelsandelectrophysiologyunder conditionsthatallowspermmembranelipidstobehaveastheydointhefemalereproductivetract.Wenext proposetodeterminetheprecisemolecularmechanismsbywhichsterolsandGM1regulateVGCC(Aim2).To dothis,we?llexpressdifferentVGCCsubunitsinspecificcelllines,aswellasartificialmembranesystems,in whichwecancontrolbothchannelexpressionandthelipidcomposition.InbothAims,wewillutilizeinnovative mousestrainseitherexpressinggeneticallyencodedCa2+indicatorsorlackingspecificchannelsubunits. Together,thesestudieswillprovidebroadmechanisticinsightintotheregulationofVGCCbymembranelipids, amatterofcriticalimportanceinbothnormalphysiologyaswellasimportantdiseasestates.